Immobilized metal affinity chromatography IMAC

The ELP expression system was compared to the conventional oligohistidme fusion, which is traditionally applied for purification by immobilized metal affinity chromatography (IMAC). Both techniques were shown to have a similar yield of the recombinant protein. The temperature-triggered approach offers a fast and inexpensive nonchromatographic separation with the possibility for larger scale purification. Although the ELP expression system may not be applicable to all types of recombinant proteins, numerous examples have already been shown [40]. 

The inhibition of SuSy by the divalent cations (fCi 15 aM), Zn (fC 25 aM), and Ni (fti 37 aM) was exploited for further purification of SuSyl by immobilized metal affinity chromatography (IMAC). A subsequent gel filtration yielded homogeneous SuSyl suitable for crystallization experiments [24]. The protein chemical characterization revealed a homotetrameric organization of the 93 kDa subunit. Our kinetic data for the cleavage reaction and preliminary immunoblot analysis for phosphoserine suggested that SuSyl may be phosphorylated in the yeast expression system [28]. 

Several scenarios can be considered with immobilized metal affinity chromatography (IMAC) systems in terms of the relative influences of these different equilibria. Thus, if Ksolvate > Kpj, the polypeptide or protein will not bind to the IMAC sorbent. Similarly, if KPl > KM + w CheL the polypeptide or protein will strip the metal ion from the immobilized chelating complex, such as observed, e.g., with native or recombinant fibroblast growth... 

This type of affinity separation is also known under the name of immobilized metal affinity chromatography (IMAC) and is based on the ion-mediated interaction with proteins. Metal ions are adsorbed first on a chelating... 

Desalting can be achieved by precipitation with ethanolic atmnonium acetate [4], solid-phase extraction (SPE), microdialysis [13], Fe " -loaded immobilized metal affinity chromatography (IMAC) [14], cation-exchange coluttms (SCX) [15-16], or combinations thereof [17-18]. The SCX procedure can be applied on-hne with direct infusion of the effluent into ESl-MS [15-16]. The mobile phase applied is similar to the one applied in LC-MS, i.e., 50% acetonitrile in 10 mmol/1 aqueous TEA. 

The foundations for immobilized metal affinity chromatography (IMAC) were first laid in 1961 when Helferich introduced ligand-exchange chromatography [1], The modern-day usage of this technique and its practical applications as a purification tool did not emerge, however, until 1975 and the seminal work by Porath et al. [2],... 

Immobilized-metal affinity chromatography (IMAC) is a separation technique that uses covalently bound chelating compounds on solid chromatographic supports to entrap metal... 

Detection of phosphorylated proteins and peptides can be enhanced by selectively isolating these species. Online immobilized metal affinity chromatography (IMAC)-CE-ESI-MS is such a powerful analytical tool. The IMAC resin retains and preconcentrates phosphorylated proteins and peptides, CE separates the phosphorylated species and MS/MS identifies the components and their phosphorylation sites. Cao and Stults applied this method to the analysis of phosphorylated angiotensin II and tryptic digests of a- and /3-casein (CE conditions buffer, 0.1% acetic acid/10% methanol uncoated capillary). Beta-casein is a well-characterized protein with five phosphorylation sites and is widely used as a standard for protein phosphorylation studies. 

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